Absorbing too little calcium (Ca) in the intestine can be a risk factor for negative Ca balance and the development of osteoporosis. An important gap in our knowledge is that the molecular determinants of Ca influx into the enterocyte and transcellular Ca transport have not been unambiguously identified. This lack of knowledge limits the development of specifically targeted therapeutic agents to manipulate Ca absorption. The long-range goal of our research program is to identify the molecular factors responsible for age-related calcium malabsorption, and to target the development of safe and effective therapeutic agents that can modulate active calcium absorption. The recent discovery of the CaT1 (also known as ECaC2 and TRPV6) gene and the demonstration that its protein product functions in non-intestinal cells as a membrane Ca transporter, and our recent report that the expression of CaT1 in the Caco-2 human intestinal cell line is regulated by 1,25-dihydroxyvitamin D, offers an exciting new opportunity to further knowledge of the molecular mechanisms of vitamin D-mediated intestinal Ca absorption. We have two specific aims in this proposal (1) to establish the role of CaT1 and calbindin D in vitamin D-mediated calcium transport and (2) to identify to what extent CaT1 expression is a modulator of the rapid non-genomic vitamin D response in the enterocyte. We will determine the functional role of these proteins in the enterocyte by using Caco-2 cells transfected with sense and antisense CaT1 and calbindin D cDNA to alter the level of these proteins independently of vitamin D. The central hypothesis of our first research aim is that CaT1 acts as the primary gatekeeper of intestinal Ca absorption by controlling, under regulation by 1,25-dihydroxyvitamin D, the rate of Ca entry across the apical brush border membrane of the enterocyte. The cytosolic vitamin D-dependent calbindin D acts in concert with CaT1 as both intracellular buffer and transporter, delivering Ca that has crossed the brush border membrane to the basolateral membrane of the enterocyte for Ca exit into the blood. We hypothesize in our second research aim that the CaT1 molecule either is responsible for or modulates the rapid 'non-genomic' response to 1,25-dihydroxyvitamin D in Caco-2 cells by determining the rate of Ca entry into the enterocyte following 1,25-dihydroxyvitamin D treatment and thereby modulating the rise of free intracellular Ca and subsequent downstream activation of protein kinase C and phospholipase D. [unreadable] [unreadable]